RPA 'Ecomed', 129090, Russia, Moscow, POB no. 87, tel. 963-11-22, tel./fax 963-05-97, email: ecomednpo@mtu-net.ru, site: www.npoecomed.com

"APPROVED"
Customer of investigations
General Director of ECOMED Research & Production Association
S.A.Khvorostov
/signature/
Seal
Date: June 01, 1995		 "APPROVED"
Director of Research Institute of Children's Hematology
Russian Federation Ministry of Health Protection
Corresponding Member of Russian Academy of Natural Sciences
Professor A.G.Rumyantsev
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R E P O R T
Antitumor properties of autonomous electrostimulator
of alimentary canal and mucosa and cytostatic preparations in cell culture


Scientific supervisor:
doctor of medical sciences,
professor		 L. G. Korkina /signature/
Responsible executor:
doctor of biological sciences,
professor		 A. M. Moroz /signature/

     The goal of this work was to study the combined effect of the autonomous electrostimulator and some cytostatic preparations on growth and reproduction of tumor cells under cultural conditions. Because cytotoxic and cytostatic effects of the electrostimulator were discovered at the first stage of investigations in the cell cultures of sarcoma and two limphoma lines, it was of interest to reveal the increase in antitumor activity of standard chemicotherapeutical preparations, which are widely applied at the oncological departments of hospitals and at specialized oncohospitals, upon their combined influence on the tumor cells in vitro. In order to estimate more comprehensively and to distinguish the effect of the electrostimulator, we studied the proliferative activity of the tumor cells, their ability to form colonies (clonogenic potential), vitality of the cells at various times of coincubation with the electrostimulator, and synergism of the effects of cytostatics and stimulator.

Materials and methods

Objects of investigation

     One kind of limphoma EL-4 tumor cells was used in this investigation. Because we studied the preparations, which are mainly applied for treatment of limphoproliferative diseases, and the volume of performed investigations was extremely great, it was considered to be expedient to use the limphoma cells to obtain reliable and comparable results. Limphoma was reinoculated to C57BL/6J(H-2) mice of 8 to 10 weeks old.

Chemical preparations

     We studied the following antitumor reparations: metatrexate (Ebene Arzneimittel, Austria) in the amounts equivalent to one therapeutical dose (3 mg/l), 0.5 therapeutical dose (1.5 mg/l), and 0.1 therapeutical dose (0.3 mg/l). Vinchrystine (Gedeon Richter, Hungary) was also used in the amounts equivalent to 1 therapeutical dose (0.1 mg/l), 0.5 therapeutical dose (0.05 mg/l), and 0.1 therapeutical dose (0.01 mg/l).

Cultivation of tumor cells

     The EL-4 cells were cultivated in 24-hole boards (Costar) in the RPMI-1640 medium containing 10% veal embryonic serum, HEPES buffer, L-glutamine, indispensable aminoacids, sodium pyruvate, antibiotics, and 2-mercaptoethanol. At various times after the beginning of cultivation, we determined the level of introduction of 3H-thymidine; on this purpose, the cells were transferred into 96-hole boards (NUNC) (50 thousand cells into each hole), added 1.5 microcurie of 3H-thymidine and cultivated for 1 hour. Then we transferred the cells to the filter using a harvester and determined the radioactivity level using a beta-counter (Beckman). We found initially that the maximum amount of 3H-thymidine is introduced into the EL-4 limphoma cells on the second day after the beginning of cultivation.

     In order to determine the antitumor activity of the electrostimulator, the limphoma cells were cultivated in 6-hole boards containing 8 ml of cultural medium with the cell concentration of (3.5-4)x105 cell/ml. The test hole contained an active electrostimulator, and the control hole contained an inactive electrostimulator as a placebo. In 1, 6, and 24 hours, the cells were resuspended directly in the holes, and some part of suspension was taken out to determine the parameters of their functioning and the structural integrity. In other series of experiments, the limphoma cells were incubated with the specified doses of chemotherapeutic preparations for 3 to 4 hours or with a combination of the electrostimulator with the chemical preparation taken in the optimum dose. After incubation, we thoroughly washed the limphoma cells from the residual chemical preparations, suspended them in the nutritious medium, and investigated their functional activity. The change in this activity enabled us to consider the antitumor properties of the agents under investigation.

Methods for determination of functioning parameters
and structural integrity of the tumor cells.

     Vitality of the tumor cells. The estimation of vitality of cells in the culture was determined from the color of a vital pigment (trypan blue); on this purpose, we added 5% solution of this pigment to the culture and in 5 minutes determined the number of colored nonviable cells using a microscope. We calculated the percentage of colorless (viable) cells in the culture.

     The proliferative activity of the tumor cells was estimated from the intensity of the DNA signal in the tumor cells that was determined by the 3H-thymidine introduction. On this purpose, we incubated 1.5 microcurie of tritium-labeled thymidine for 1 hour, transferred the cells on the filter, and determined the radioactivity level on the filter. The results were expressed in the form of the depression index of 3H-thymidine introduction I, which is the ratio of the number of pulses in the test culture to the number of pulses in the control culture. Each point was measured in triplication. We performed five independent experiments. The statistical treatment of results was carried out using the Student method with 5% criterion of reliability of difference between the test and the control results.

     Colonization function of the tumor cells. The estimation of clonogenic activity of the tumor cells was carried out using boron silicate capillaries. The tumor cells in the amount of 500000 cell/ml in the whole medium of RPMI 1640 with 10% veal embryonic serum were mixed with agar prepared with twice distilled water and placed into a capillary. The capillaries were incubated for 14 days at 37 C in the atmosphere containing 5% of carbon dioxide. The clusters containing at least 50 tumor cells were considered as colonies. The efficiency of colonization was estimated as the percentage of colonies with respect to the number of cells sown. Three capillaries were filled for each point. The statistical analysis was performed using the Student method.

     Activity of intracellar dehydrogenases. The working activity of the cell enzymes was estimated from the intensity of introduction and reduction of tetrazolium salts (this method was first suggested by T.Mossman in 1983 [1]). The method is based on investigation of activity of some dehydrogenases in mitohondria of living actively functioning cells to reduce tetrazolium salts, in particular, MTT bromide. Meanwhile, the light blue water-soluble substrate (MT) is transformed into dark blue formazane crystals, which are insoluble in water. The reaction intensity is quantitatively measured using the colorimetric method. The amount of formazane formed is used as a criterion of redox enzymatic reactions in the living cells that enables to determine the degree of damage of the tumor cells under the influence of various agents [2, 3]. The tumor cells after incubation with the electrostimulator, chemical preparation, or combination thereof, were dropped in triplet using 96-hole boards (Costar) in the amount of 50000 cells in the volume of 100 microliters per a hole. We added 25 microliters of MTT (Sigma) in physiological solution (5 mg/l) into each hole. Then, after three-hour incubation at 37 C in the atmosphere containing 5% of carbon dioxide, we added 150 microliters of isopropanol into each hole and centrifuged at 3000 c.p.m. for 10 min. Finally, we sampled 100 microliters of liquid over the precipitate from each hole, dropped this liquid in clean holes of a 96-hole board, and determined the color indicator using a multiscan (MCC 340 P, Lab System) at the wavelength of 540 nm. The statistical analysis of differences between the control and test holes was carried out using the Student method.

Results

1. Vitality of cells in the culture.

     The number of viable cells in the EL-4 limphoma culture was 91.9(3.6)% in average. In four hours of incubation, the number of viable cells in the culture did not virtually change and was 89.2(2.1)%. The corresponding parameter in the test cultures was as follows:
in the cultures with the electrostimulator - 88.2(1.9)%
in the cultures with metatrexate - 85.6(2.7)%
in the cultures with vinchrystine - 86.1(2.3)%
in the cultures with the combination of electrostimulator and metatrexate - 83.9(2.0)%
in the cultures with the combination of electrostimulator and vinchrystine - 82.9(3.1)%

     Upon statistical treatment of the results, we found that in all the cases the differences between the control and test cultures were uncertain (p > 0.05).

     Thus, incubation of the EL-4 limphoma cells with the electrostimulator or chemical preparations, or combination of these agents results in an insignificant decrease in the vitality of cells in the culture. Therefore, neither the preparations under investigation nor the electrostimulator affect the rate of necrotic processes in the culture of tumor cells; i. e., they do not change essentially the penetrability of the cell membranes for ions and water

2. Change in the rate of proliferation of tumor cells in the culture.

     In order to determine the proliferative activity of tumor cells in the culture, we used a modification of the standard method to adapt it to the conditions of influence of weak electromagnetic fields generated by the electrostimulator. In brief, the essence of this modification was as follows: after incubation of cells with the electrostimulator, medicines, or their combination for 4 hours, the cells were washed and transferred into 24-hole boards (Nunc) for further cultivation for 24 hours. Then we transferred the limphoma cells into 96-hole boards (100000 cells/hole), added 3H-thymidine in the amount of 1 microcurie per a hole and incubated for 1 hour. After the incubation was completed, we measured radioactivity in the washed cells and calculated the depression index of introduction of 3H-thymidine, which is the precursor in the DNA synthesis. The results were expressed as the average value of data obtained in three independent experiments with consideration of the standard deviation value. The results of experimental data are represented in Table 1. We can see that incubation of the tumor cells with the autonomous electrostimulator resulted in depression of introduction of the radioactive label into DNA. Virtually the same results were obtained upon determination of the depression index immediately after termination of the influence of a weak electromagnetic field and in 24 hours after termination of its influence. On the basis of these experimental results, we could conclude that the distant inhibiting effect of electromagnetic field is retained in the culture of viable tumor cells at least for one day.

     Investigation of the influence of metatrexate and vinchrystine on depression of proliferation of the limphoma cells immediately after termination of the influence demonstrated the absolute absence of the effect: the cells continued to synthesize DNA at the former rate. The combined influence of the electrostimulator and chemical preparations taken even in maximum doses revealed that immediately after termination of incubation all the observed effect of inhibition of proliferation depended upon the presence of the stimulator in the medium for cell incubation.

     The inhibiting influence of chemical preparations became expressed only in 24 hours after the incubation was completed. Meanwhile, metatrexate and vinchrystine taken in the doses of 1 and 0.5 of the therapeutical values almost completely depressed the synthesis of DNA in the tumor cells that indicated 100% death of the limphoma cells. It is interesting that the death of the limphoma cells under the influence of chemical preparations was primarily apoptotic rather than necrotic. Under these conditions of the virtually complete death of cell elements in the culture, it seemed to be impossible to estimate the synergistic effect of the stimulator and chemotherapeutic preparations. However, upon reduction of the dose of chemotherapeutic preparations in the culture to 0.1 of the therapeutical dose, the depression index of proliferation was significantly lower (Table 1). Meanwhile, the combined influence of a weak electromagnetic field and the preparation appeared to be significantly higher than that observed upon the individual influence of the studied inhibitors of the DNA synthesis (Table 1). The level of depression of introduction of labeled thymidine upon the combined influence of preparations and the electrostimulator was virtually equal to that found upon the influence of preparations taken in essentially larger amounts (1 and 0.5 of the therapeutical doses).

Table 1. Depression of 3H-thymidine introduction upon incubation with the electrostimulator and chemical preparations
Influence Dose and time of influence (hours) Introduction of labeled thymidine
- - 1.0
Electrostimulator 4 hours 0.63 (0.015)
Metatrexate 1 th. dose, 24 h 0.022 (0.008)
Metatrexate 0.5 th. doses, 24 h 0.035 (0.009)
Metatrexate 0.1 th. doses, 24 h 0.14 (0.03)
Electrostimulator+metatrexate 0.1 th. doses, 24 h 0.048 (0.007)*
Vinchrystine 1 th. dose, 24 h 0.018 (0.001)
Vinchrystine 0.5 th. dose, 24 h 0.03 (0.007)
Vinchrystine 0.1 th. dose, 24 h 0.13 (0.03)
Electrostimulator+vinchrystine 0.1 th. dose, 24 h 0.05 (0.01)*
*P<0.05 with respect to the influence of the single preparation

3. Colonization ability of the tumor cells.

     The initial ability of the limphoma cells to form colonies in the culture was 20.3(2.1), and the efficiency of colonization (EC) was 0.004%. After 4-hour incubation of the limphoma cells with the electrostimulator, the number of colonies reliably and significantly reduced to 8.0(0.6), and EC was 0.0016%. The same level of reduction was observed upon incubation with vinchrystine: the number of colonies was 7.0(0.6) and 8.0(0.9) and EC was 0.0014 and 0.0016% for 1 and 0.1 therapeutical doses of vinchrystine. Upon incubation with metatrexate, the effect of inhibition was significantly lower: the number of colonies was 16.0(1.4) and 16.0(1.3) and EC was 0.0032 and 0.0032% for 1 and 0.1 therapeutical doses of metatrexate. The combined influence of the electrostimulator and chemical preparations did not result in further enhancement in the inhibiting activity on the colonization process in the culture of limphoma cells (the number of colonies in the presence of the electrostimulator and vincrhystine was 8.0(0.5) and EC was 0.0016%. The number of colonies in the presence of the electrostimulator and metatrexate was 7.0(0.6) and EC was 0.0014%.

     On the basis of the results obtained in this series of experiments, we can conclude that a weak electromagnetic field generated by the electrostimulator inhibits the colonization activity of the tumor cells in limphoma at the level of effect of vinchrystine taken in a higher concentration equivalent to 1 therapeutical dose. At the same time, the electrostimulator is a significantly more efficient inhibitor of tumor colonization in the culture as compared to metatrexate. Due to the high inhibiting activity of the autonomous electrostimulator, it was impossible to demonstrate its synergistic effect with chemical preparations. Inhibition of colonization of tumor cells in the culture is a good prognostic indicator for inhibition of tumor metastasis in an organism.

     Analysis of the parameters obtained using the MTT test revealed the following regularities. While the color indicator of tetrazolium introduction into the tumor cells before any influences was 0.312(0.012), after 4-hour incubation with the electrostimulator this parameter significantly decreased to 0.150(0.012). This reduction was 52% of the control value. The same value of the color indicator was obtained after incubation of the cells in the presence of vinchrystine taken in the maximum concentration: 0.153(0.0006), i.e., 51% of the control value. The combined influence of the electrostimulator and vinchrystine did not result in the further depression of the dehydrogenase activity: 0.149(0.0009), i.e., 54% of the control value. Metatrexate was a significantly weaker inhibitor of metabolic activity of the tumor cells, because after incubation with metatrexate the color indicator decreased only to 0.257(0.020) that was 82% of the control value for the metatrexate concentration equivalent to 0.1 therapeutical doses. Upon the increase in the metatrexate concentration up to 1 therapeutical dose, the degree of depression of dehydrogenase did not change: 0.243(0.012), i. e., 77.8% of the control value. At the same time, the combined influence of metatrexate and the electrostimulator resulted in the reliable and significant enhancement in inhibition of the enzymatic activity: 0.135(0.005), i. e., 57% of the control value, p < 0.05.

Conclusion

     On the basis of the results obtained we can make some conclusions an recommendations, which are of both scientific and practical interest:
  1. We obtained new experimental evidence of the inhibiting effect of weak electromagnetic field generated by the autonomous electrostumulator on the growth of tumor cells in the culture. Meanwhile, the studied physical factor affects the DNA synthesis of the tumor cells, activates the apoptotic death of these cells, and does not virtually affect the intensity of necrotic processes in the limphoma cells. We noted a significant synergistic effect of the electrostimulator with such chemical preparations as vinchrystine and metatrexate, which are widely applied the the tumor therapy.

  2. The autonomous electrostimulator of alimentary canal and mucosa essentially inhibited the colonization activity of the tumor cells; meanwhile, its efficiency was equivalent to that obtained under the influence of high concentrations of vinchrystine rather than metatrexate. An extremely high inhibiting activity of the electrostimulator did not enable us to demonstrate the synergistic effect in combination with chemical preparations.

  3. The electrostimulator appeared to be a highly efficient inhibitor of metabolic activity of the tumor cells that is determined by the activity of mitohondrial dehydrogenase. Its inhibiting effect was comparable with the efficiency of vinchrystine taken in the maximum concentration. In this case, we observed a good synergistic effect of the stimulator and chemical preparations.

  4. On the whole, we can conclude that the electrostimulator results in significant structural and functional damages in the limphoma cells. The mechanism of inhibiting influence of the stimulator is similar to the mechanism of vinchrystine effect. Moreover, the combined application of the electrostimulator with such chemical preparations as vinchrystine and metatrexate significantly strengthens the tumorcidal properties of the chemical preparations, so application of small doses of preparations with the electrostimulator produces the same effect as the influence of preparations taken in maximum doses. This fact enables us to assume the possibility of combined application of the electrostimulator with highly toxic chemical preparations, which dosage can be significantly reduced without the loss of the desired therapeutical effect. However, the latter conclusion is preliminary and requires additional evidences and comprehensive verification. It seems to be interesting to analyze the possible application of the electrostimulator with other antitumor chemical preparations.

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